Schwann cell glycogen selectively supports myelinated axon function

Abstract

Objective: Interruption of energy supply to peripheral axons is a cause of axon loss. We determined whether glycogen was present in mammalian peripheral nerve, and whether it supported axon conduction during aglycemia.

Methods: We used biochemical assay and electron microscopy to determine the presence of glycogen, and electrophysiology to monitor axon function.

Results: Glycogen was present in sciatic nerve, its concentration varying directly with ambient glucose. Electron microscopy detected glycogen granules primarily in myelinating Schwann cell cytoplasm, and these diminished after exposure to aglycemia. During aglycemia, conduction failure in large myelinated axons (A fibers) mirrored the time course of glycogen loss. Latency to compound action potential (CAP) failure was directly related to nerve glycogen content at aglycemia onset. Glycogen did not benefit the function of slow-conducting, small-diameter unmyelinated axons (C fibers) during aglycemia. Blocking glycogen breakdown pharmacologically accelerated CAP failure during aglycemia in A fibers, but not in C fibers. Lactate was as effective as glucose in supporting sciatic nerve function, and was continuously released into the extracellular space in the presence of glucose and fell rapidly during aglycemia.

Interpretation: Our findings indicated that glycogen is present in peripheral nerve, primarily in myelinating Schwann cells, and exclusively supports large-diameter, myelinated axon conduction during aglycemia. Available evidence suggests that peripheral nerve glycogen breaks down during aglycemia and is passed, probably as lactate, to myelinated axons to support function. Unmyelinated axons are not protected by glycogen and are more vulnerable to dysfunction during periods of hypoglycemia. .

Figure 1

Glycogen content of sciatic nerves. A. Nerves bathed for 2 hours in 2, 4, 7, 10 or 30 mM glucose showed increasing concentrations of glycogen (open squares: left axis - pmoles µg protein⁻¹). Sciatic nerves removed from mice, with a mean blood glucose concentration of 9.5 ± 1.8 mM at the time of death, and immediately frozen for glycogen assay had a glycogen content of 8.61 ± 0.90 pmoles µg protein⁻¹ (grey square). B. [Glucose] plotted on a logarithmic scale demonstrating the exponential relation between glycogen content and ambient [glucose]. Left axis as main figure. The open circles and dotted line denote historical data from the mouse optic nerve , for comparison.

Figure 2

Glycogen granules are present in Schwann cell cytoplasm of A fibers. A & C. Low power image reveals glycogen granules present in the cytoplasm. B. Higher power image of the cytoplasm reveals multiple glycogen granules visualized as dark spherical granules located throughout the cytoplasm (g). Ribosomes attached to endoplasmic reticulum (ER) are also present. D. Nerves incubated in aglycemic conditions showed a decreased density of glycogen granules. A & D Scale bar 500 nm. C same magnification as D. B Scale bar 100 nm.

Figure 3

Expression of glycogen phosphorylase in sciatic nerve. A - D. Immunohistochemical studies revealed the presence of glycogen phosphorylase (green), whose cellular location was identified using specific cellular markers for Schwann cells (S-100; red) or axons (neurofilament; blue). D. Merged images demonstrate that glycogen phosphorylase is detected in both Schwann cell cytoplasm (yellow) and axons (cyan). Scale bar 10 µm.

Figure 4

The compound action potential (CAP) recorded from CD-1 mouse sciatic nerve. A. Stimulus evoked CAP illustrates the large rapid A fiber response immediately after the stimulus artifact (*). Scale bars 0.5 ms and 2 mV. B. The A peak is followed by the slower and smaller C fiber response approximately 10 ms after the stimulus artifact (inset). Scale bars 1 mV and 1 ms: inset scale bars 0.05 mV and 1 ms. C. Increasing the stimulus current results in increased amplitude of the A (1) and C (2) fiber response, respectively. Stimulus artifact in 1 removed for clarity. Scale bars 5 mV and 0.25 ms for 1 and 0.25 mV and 1 ms for 2. D. Plotting the stimulus intensity versus the normalized amplitude of the A (□) and C (△) fiber CAPs, demonstrates the lower threshold for activation of the A fibers relative to the C fibers response. E. Aglycemia and glycogen content of sciatic nerve. Aglycemia, indicated by horizontal bar, resulted in onset of delayed failure of the A peak after about 2 hours, and total failure after about 4.5 hours (□), whereas the C peak response failed after about 0.5 hours and disappeared by 3 hours (◇). Glycogen content (open columns; right axis) decreased during aglycemia reaching a nadir after about 2 hours, coincident with the onset of A peak failure.

Figure 5

Glycogen content of sciatic nerves and latency to CAP failure during aglycemia. A. A fiber peak in nerves pre-incubated in 2 mM, 10 mM or 30 mM glucose for 2 hours prior to onset of aglycemia at 0 hrs. The peak amplitude was normalized to the value at aglycemia onset. The response was maintained longest in nerves pre-incubated in 30 mM glucose (○), followed by pre-incubation in 10 mM (□) then 2 mM (△) glucose. Horizontal bar indicates aglycemia – also applies to B. B. The latency to C fiber peak failure during aglycemia showed the same pattern of failure as the A fiber response, although the latencies were shorter. Symbols for A also apply. C. Relationship between latency to 95% CAP failure and glycogen content at the onset of aglycemia of the A (□) peak, demonstrating a steep linear relationship. The relationship between latency to 95% CAP failure and glycogen content at the onset of aglycemia of the C (○) peak is not as steep as that of the A peak. D. A comparison of the latency to failure of C fibers pre-incubated in 10 mM glucose (○) and A fibers pre-incubated in 2 (△) or 10 mM (□) glucose prior to aglycemia highlights the beneficial effect of glycogen on A fiber conduction. Horizontal bar indicates aglycemia.

Figure 6

Effect of inhibiting glycogen phosphorylase on latency to CAP failure and glycogen content. A. Introduction of the glycogen phosphorylase inhibitor DAB resulted in accelerated failure of the A fiber peak (○) relative to control, untreated nerves (□). Horizontal bar indicates aglycemia – applies to B and C. B and C. DAB had no effect on latency to CAP failure of C fibers incubated in 10 mM (B) or 30 mM (C) glucose. D. DAB significantly accelerated CAP failure after onset of aglycemia in A, but not C fibers incubated in 10 mM glucose or 30 mM glucose. E. DAB increased glycogen content in sciatic nerves incubated in 10 mM glucose compared to nerves incubated in 10 mM glucose without DAB (left-hand columns). After 2 hours of aglycemia (filled columns) the glycogen content was elevated in nerves incubated in DAB compared to nerves incubated in the absence of DAB: ns = not significant; * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0005.

Figure 7

Lactate is equivalent to glucose in supporting sciatic nerve function. A. In the presence of 10 mM glucose (□) or 20 mM lactate (◇) the A fiber CAP is stable for hours. B. In the absence of glucose (from time ‘0’), the A fiber CAP begins to fail after about 2 hours (◇). If glucose (□) or lactate (◇) is introduced after 2.5 hours of aglycemia, when the CAP had fallen to about 50% of baseline value, the CAP recovers fully. C. In the presence of 10 mM glucose (□) the C fiber CAP is stable for hours, but in the presence of 20 mM lactate (◇) the C fiber CAP amplitude gradually decreases. D. In the absence of glucose the C fiber CAP begins to fail after about 1 hour (◇). If glucose (□) or lactate (◇) is introduced after about 2 hours of aglycemia, when the CAP had fallen to about 50% of baseline value, the C fiber CAP (◇) only partially recovers.

Figure 8

Lactate is present in the extracellular space. A. An enzyme electrode was used to directly measure extracellular lactate concentration, [lac⁻]_o. The active sensor (grey) of the enzyme electrode was pressed alongside the optic nerve, while simultaneously recording the stimulus evoked CAP via suction electrodes. B. The [lac⁻]_o was zero in the bath (not shown) but about 120 µM at the edge of the sciatic nerve. After about 10 min of aglycemia the [lac⁻]_o begins to fall steeply and reaches nearly zero after 20 min. The response of the A (□) and C (◇) fibers has included for temporal comparison. C. Introduction of DAB (horizontal bar) results in a reversible fall of about 35% in the lactate signal.